title: Capillary electrophoresis for analysis of deletion and duplication in exon 44-55 of Duchenne muscular dystrophy gene.
abstract: In this study, a genotyping CGE method was established for analysis of Duchenne muscular dystrophy (DMD) gene deletions and duplications in exon 44-55. A total of 12 DMD exons (exon 44-55) and 2 internal standard gene fragments were simultaneously amplified by using a universal multiplex PCR (UMPCR) and determined by CGE. The conditions of UMPCR and CGE were optimized, including the kinds of polymerase, temperatures in UMPCR, separation matrix, separation temperature, and voltage. Finally, the separation was performed by 1.2% poly(ethylene oxide) in 1× TBE buffer at -6 kV and 25°C. After validation, our results showed the peak patterns for differentiation of genetic deletion or duplication in 27 DMD patients and normal subjects, according to the peak height ratios by comparison of two internal standard peaks. Among the 27 subjects, 23 cases are deletion type and four are duplication type. The data of two patients analyzed by this CGE-PCR method were different from that of multiplex ligation dependent probe amplification method, and the sequencing results demonstrated that our results were correct. This UMPCR-CGE method was considered better than the multiplex ligation dependent probe amplification method. Furthermore, this method can be used for eugenics in clinical applications.
authors.affiliation: School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Chung-An
authors.initials: CA
authors.lastname: Chen
authors.forename: Ming-Yuh
authors.initials: MY
authors.lastname: Chang
authors.forename: Tung-Ming
authors.initials: TM
authors.lastname: Chang
authors.forename: Yuh-Jyh
authors.initials: YJ
authors.lastname: Jong
authors.forename: Shou-Mei
authors.initials: SM
authors.lastname: Wu
mesh_terms: D016022:Case-Control Studies; D004252:DNA Mutational Analysis / Q000379:methods*; D016189:Dystrophin / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005091:Exons; D005452:Fluoresceins / Q000737:chemistry; D017353:Gene Deletion; D020440:Gene Duplication; D006801:Humans; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics*
chemical_list: D016189:Dystrophin; D005452:Fluoresceins; C024098:6-carboxyfluorescein
keywords: Capillary gel electrophoresis; Duchenne muscular dystrophy; Genotyping